PMC

(a) Average tumor volume for involution group (I) and involution+CXB group (I + CXB) mice at 3 weeks post-injection, p=0.0205, n=12, unpaired t test. (b) % glands with dispersed GFP+ tumor cells in mammary glands at 3 d post-injection, p=0.018, n=12, unpaired t test. (c) % glands with dispersed GFP+ tumor cells in mammary glands at 3 weeks post-injection, p=0.0033, n=12, unpaired t test. Data are presented as mean +/− SEM. (d) SHG imaging of collagen surrounding control involuting (Inv)and CXB treated involuting ducts (Inv + CXB), scalebar=50 µm. (e) Collagen intensity by SHG versus distance from involuting mouse mammary ducts (red, n=21 ducts, 3 mice) compared to involuting + CXB (green, n=22 ducts, 3 mice) and to nulliparous (black, n=12 ducts, 3 mice), p<0.001 Involution vs Involution + CXB between 5–10 um, student’s t test. (f)A model depicting COX-2, derived from the involuting mammary gland, mediated upregulation of collagen fibrillogenesis and subsequent upregulation of COX-2 and invasion (brown cells) in tumor cells exposed to involution collagen.

(a) Trichrome (top) and COX-2 (bottom) IHC-stained tumor, arrows=dense collagen, arrowhead=sparse collagen, scalebar=50 µm. (b) Right, percent tumor COX-2+ by IHC, *p=0.0275, n=9 (nulliparous), n=5 (involution), unpaired t test Left, COX-2 IHC images, scalebar=100 µm. (c) Western blot showing expression of COX-2 in tumor cell populations, *p=0.0274, n=3 per group, unpaired t test. (d) In vitro wound closure by tumor cells on collagen +/− 20 µM COX-2 inhibitor celecoxib (CXB, black) at 6h post-scrape, *p=0.019, n=3 cell populations per group, unpaired t test. Inset: individual tumor cell population data. (e) Left, % COX-2+ organoids by IHC in Matrigel™ or Matrigel™ + 10% (10), 20% (20), and 40% (40) collagen, *p<0.0001, one-way ANOVA. Right, 3D-organoid COX-2 IHC images, scalebar=50 µm. (f,g,h) % organoids rounded(1), dysmorphic(2), or invasive(3) on (f) 0 and 40% collagen, *p<0.0001, n=3 wells per condition, one way ANOVA, (g) on 40C + DMSO solvent, 40C + 2.5 µm CXB, 40C + 5 µm CXB, *p<0.0001, n=3 wells per condition, one way ANOVA and (h) on 40C + shGFP and 40C + shCOX-2, *p<0.0001, n=3 wells per condition, one way ANOVA. (i) Left, SHG image of collagen surrounding nulliparous and involuting ducts, higher magnifications (yellow box) below, scalebar=10 µm. Arrow: radially aligned collagen, scalebar=50 µm. (j) Organoid type on 0C, 20% collagen + 20% gelatin (20/20), 40C and 40% gelatin (40G), *p<0.0001, n=3 wells per condition, one way ANOVA. Data are mean +/− SEM and representative of triplicate studies.

(a) In vitro wound closure untreated (U), vehicle treated (V), and ibuprofen treated (200µg mL⁻¹) (Ibu), *p=0.0001, n=4 wells per condition, unpaired t test. (b) Collagen quantification by SHG as intensity versus distance from rat mammary ducts, black=nulliparous, red=involution, green = involution + ibuprofen (3 rats per Group) p<0.001, involution v involution+ibuprofen between 5–10 um from duct, student’s t test. (c) H&E images of involution and involution+ibuprofen group mouse mammary tissues 2 weeks post-treatment, top scalebars=150 µm, bottom scalebars=40 µm. (d) Average tumor volume in nulliparous (N), involution (I), +/− ibuprofen (Ibu) group mice, *p=0.00373, one way ANOVA, ‡p≤0.035, type IIIF test for group effect in additive model, n=6 (nulliparous), n=8 (involution), n=4 (nulliparous + ibuprofen), n=5 (involution + ibuprofen). (e) Tumor burden, 6 week timepoint, *p=0.0255, n=8 (involution), n=9 (involution+ibuprofen), unpaired t test. (f) Quantitation of tumor COX-2 expression at 6 week timepoint, *p≤0.036, unpaired t test, n=9 (nulliparous), n=9 (involution), n=6 (nulliparous+ibuprofen), n=9 (involution+ibuprofen). (g) Statistical modeling of mouse lung signal for human-specific β2M by qRT-PCR across time, ‡p=0.027, t test of group effect. (h) Left axis, average mammary tumor volume per Group demonstrating size-match between groups, (light gray). Right axis, mouse lung signal for human-specific β2M by qRT-PCR analysis, *p=0.011, n=5, Wilcoxon test, involution=dark gray, involution+ibuprofen=black. Data are mean +/− SEM.

(a) Average primary tumor volume, *p=0.001, **p=0.0174, n=7 (nulliparous), n=6 (involution), unpaired t test. (b) Total tumor number per group at 4 weeks post-injection, n=9 (nulliparous) and n=8 (involution). (c) Average tumor burden (total tumor volume per injected gland) 4 weeks post-injection, *p=0.0011, n=15 (nulliparous), n=13 (involution), unpaired t test. (d) Average percent tumor area positive for Ki67, *p=0.0009, n=5 (nulliparous), n=8 (involution), unpaired t test. (e) Collagen intensity measured by Second Harmonic Generation (SHG) imaging versus distance from involuting mouse mammary ducts (red, n=17 ducts, 3 mice) compared to nulliparous (black, n=12 ducts, 3 mice), p<0.00001, student’s t test. (f) Western blot for Collagen I in mouse mammary tissue lysates, *p=0.042, n=3 per group, unpaired t test. (g) Percent tumor area positive for collagen by trichrome stain, *p=0.0002, n=6 (nulliparous), n=11 (involution), unpaired t test. (h) Top, trichrome stained tumor images, blue=collagen. Bottom, blue signal converted to black, scalebar=100 µm. (i) Percent BRDU+ cells in 3D culture on Matrigel or 10% collagen I, *p<0.0001, unpaired t test, n=30 100X fields per group. (j) BRDU IHC images of 3D organoids, scalebar=50 µm. N=nullip=nulliparous (white), I=inv=involution (gray), data are presented as mean +/− SEM.

(a) GFP+ tumor cells 3 weeks post injection, scalebar=100 µm. (b) IHC image of GFP+ cells in mammary tissue 3 d post-injection, scalebar=50 µm, inset GFP+ tumor cells, scalebar=10 µm, and quantification of dispersed GFP+ cell clusters by group, *p=0.0129, n=3 (nulliparous), n=5 (involution), unpaired t test. (c) IHC image of tumor cells in mammary blood vessel 3 d post-injection, scalebar=10 µm. Quantification of tumor cells in peripheral blood 1 and 3 d post-injection, *p=0.04, n=3 per group, unpaired t. (d) Fluorescent in situ hybridization for COT-1 DNA (human=red, mouse=green) and DAPI-stained nuclei (blue), scalebar=50 µm. (e) Left axis, individual mammary tumor volumes (black bars). Right axis, qRT-PCR analysis of lung for human β2M transcripts in arbitrary units (a.u.) after normalizing to actin. Inset, average β2M expression *p=0.0046, n=9 (nulliparous #1-9), n=8 (involution #10-17), unpaired t test. (f) Right, average in vitro wound closure of Involution and Nulliparous Group tumor cell populations on collagen 2, 4, and 6 h post-scrape *p=0.034, **p=0.045, ***p=0.040, n=7 (nulliparous), n=8 (involution). Left, scrape images of Involution Group tumor cell populations at 0 h and 6 h (scalebar=50 µm). (g) Left, average number of invasive tumor cells in transwell assay, n=5 (nulliparous), n=6 (involution), *p=0.0491 unpaired t-test. Right, filter images, scalebar=50 µm. (h) Left axis, volumes of tumors utilized for tumor cell isolation (black bars). Right axis, wound closure of individual tumor cell populations 6h post-scrape. N=nullip=nulliparous (white), I=inv=involution (gray), data are mean +/− SEM.

(a) Quantification of collagen intensity by SHG versus distance from human breast ducts, black=nulliparous, red=involution (13–14 ducts per case, 3 cases per group), p<0.00001, student’s t test. Data are presented as mean +/− SEM. (b) SHG imaging of collagen in breast tissue from involuting (inv) and nulliparous (nullip) women, scalebar=50 µm. (c) Multivariate Cox Analysis of relapse free survival in 345 breast tumors diagnosed in women ≤45 years of age who relapsed within five years of diagnosis for effect of high Col1A1 and COX-2 (gene name: PTGS2) expression (23%) (1-red dashed line), all other combinations of Col1A1 and COX-2 expression (77%) (0-blue line), and ER status (58% pos and 42% neg). High Col1A1 and COX-2 is the only significant variable p=0.018. Univariate analysis of (d) high COX-2 and (e) high COL1A1 and relapse free survival in 345 breast tumors diagnosed in women ≤45 years of age who relapsed within five years of diagnosis. (f) Left, images of human DCIS lesions stained for COX-2 by IHC, scalebar=50 µm, * normal adjacent tissue. (g) percent area positive for COX-2 signal quantitated by quantitative IHC methods, p= 0.0266 n=11 nulliparous cases and 22 cases diagnosed ≤ 10 yrs postpartum (2–13 DCIS lesions examined per case), unpaired t-test. See for data set characteristics. Nullip=nulliparous (white), PP=postpartum (gray), black bars indicate group average.